Reducing the immunogenicity of anti-CD20 antibodies by framework patching

ABSTRACT

Framework (FR)-patching is a novel approach to modify immunoglobulin for reducing potential immunogenicity without significant alterations in specificity and affinity. Unlike previous described methods of humanization, which graft CDRs from a donor onto the frameworks of a single acceptor immunoglobulin, we patch segments of framework (FR 1 , FR 2 , FR 3 , and FR 4 ), or FRs, to replace the corresponding FRs of the parent immunoglobulin. Free assortment of these FRs from different immunoglobulins and from different species can be mixed and matched into forming the final immunoglobulin chain. A set of criteria in the choice of these FRs to minimize or eliminate the need to reintroduce framework amino acids from the parent immunoglobulin for patching is described. The approach gives greater flexibility in the choice of framework sequences, minimizes the need to include parent framework amino acids, and, most importantly, reduces the chances of creating new T- and B-cell epitopes in the resultant immunoglobulin.

This application is a divisional of U.S. application Ser. No.09/892,613, filed on Jun. 27, 2001, now U.S. Pat. 7,321,026, thecontents of which are hereby incorporated by reference into thisapplication.

Throughout this application, various references are referred to withinparenthesis. Disclosures of these publications in their entireties arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains.

FIELD OF THE INVENTION

The present invention relates to novel methods in re-engineering, orreshaping antibodies with clinical indications (both therapeutic anddiagnostic). The method combines the use of recombinant technology and,stepwise and systemic approaches in re-designing antibody sequences. Theinvention particularly provides for antibodies which are modified to beless immunogenic than the unmodified counterpart when used in vivo.

BACKGROUND OF THE INVENTION

Monoclonal antibodies (Mabs) have become the most successful proteindrugs being used for the treatment of a variety of diseases, includingcancers, transplantation, viral infection, etc. However, the concept ofmagic bullet took more than 25 years to realize, because there wereproblems associated with the use of monoclonal antibodies. One of themain problems stems from the original source of most monoclonalantibodies, which are of rodent and murine origin. Repeated injectionsof these foreign proteins into human would inevitably result in theelicitation of host immune responses against the antibodies: theso-called human anti-mouse antibody (HAMA) responses. Although earlierattempts to use the techniques of molecular engineering to constructchimeric antibodies (for example, mouse variable regions joined to humanconstant regions) were somewhat effective in mitigating HAMA responses,there remains a large stretch of murine variable sequences constituting⅓ of the total antibody sequence that could be sufficiently immunogenicin eliciting human anti-chimeric antibody (HACA) responses. A moreadvanced improvement in antibody engineering has recently been utilizedto generate humanized antibodies in which the complementaritydetermining regions (CDR's) from a donor mouse or rat immunoglobulin aregrafted onto human framework regions (for example, EPO Publication No.0239400, incorporated herein by reference). The process is called“humanization”, or “CDR-grafting”. The original concept of humanizationdescribes the direct grafting of CDR's onto human frameworks, reducingthe non-human sequences to less than 5%, and thereby the HAMA and HACAresponses. However, direct replacement of framework sequences withoutfurther modifications can result in the loss of affinity for theantigen, sometimes as much as 10-fold or more (Jones et al., Nature,321:522-525, 1986; Verhoyen et al., Science, 239:1534-1536, 1988). Tomaintain the affinity of the CDR-grafted or humanized antibody,substitutions of a human framework amino acid of the acceptorimmunoglobulin with the corresponding amino acid from a donorimmunoglobulin at selected positions will be required. The positionswhere the substitution takes place are determined by a set of publishedcriteria (U.S. Pat. No. 5,85,089; U.S. Pat. No. 5,693,762; U.S. Pat. No.5,693,761; incorporated herein by reference). However, the presence ofmurine amino acids within stretches of human framework sequences can beimmunogenic in the generation of new T- and B-cell epitopes. Moreover,the identification of the proper framework amino acids to be replacedcan sometimes be difficult, further reducing the chances of success inhumanization without significant impacts on the specificity and affinityof the humanized antibody.

New and improved means for producing re-engineered immunoglobulin withreduced or eliminated immunogenicity while maintaining the specificityand affinity of the parent antibody are therefore needed. Preferably,the re-engineered immunoglobulin should contain no FR amino acidsubstitutions from the parent antibody, which can be a likely source ofimmunogenic epitopes for T- or B-cells. However, the approach alsooffers flexibility in the sequence design where few murine residues or astretch of murine sequences can be included in the final design, withthe ultimate goal of reducing immunogenicity while maintainingspecificity and affinity of the resultant antibody for human uses. Thepresent invention describes the methods and approaches in fulfillingthese goals.

SUMMARY OF THE INVENTION

The present invention relates to novel methods for re-engineeringimmunoglobulin chains having generally one or more complementaritydetermining regions (CDR's) from a donor immunoglobulin and portions offramework sequences from one or more human, or primate immunoglobulins.The preferred methods comprise first dividing the framework sequencesfrom immunoglobulins of all species into compartmentalized subregions ofFR1, FR2, FR3, and FR4, according to the Kabat Database (Kabat et al.Sequences of proteins of immunological interest. Maryland: US Departmentof Health and Human Services, NIH, 1991), and comparing the individualFR's, instead of the whole framework, in the variable region amino acidsequence subregions of the parent immunoglobulin to correspondingsequences in a collection of human, or primate immunoglobulin chains,and selecting the appropriate human or primate FR's with the highestdegree of homology to replace the original FR's of the parentimmunoglobulin (framework- or FR-patching). The human FR's can beselected from more than one human or primate immunoglobulin sequences. Acollection of human or primate immunoglobulin sequences can be obtainedfrom different databases (for example, Kabat database, NationalBiomedical Research Foundation Protein Identification Resource,Brookhaven Protein Data Bank, internet, etc.). The individual FRsequences selected from human or primate immunoglobulins will typicallyhave more than 60% homology to the corresponding parent FR sequences.Although high overall homology will be an important criteria forselecting the FR's for patching, lesser homology FR's will be used ifthe homology of sequences directly flanking the CDR's or at looppositions where contact(s) with the antigen binding site is (are)determined experimentally or predicted via computer modeling. The parentimmunoglobulin whose FR's are to be patched may be either a heavy chainor light chain. A patched light and heavy chain can be used to form acomplete FR-patched immunoglobulin or antibody, having two light/heavychain pairs, with or without partial or full-length human constantregions.

The individual FR's chosen for patching a parent immunoglobulin chain(applies to both heavy and light chains) should:

-   -   (1) preferably have amino acid sequences immediately adjacent to        the CDR's identical to that of the parent immunoglobulin chain;    -   (2) have amino acid sequences immediately adjacent to the CDR's        conservatively similar in structure to, if not completely        identical to, that of the parent immunoglobulin chain;    -   (3) preferably have identical amino acid at corresponding FR        position of the parent immunoglobulin predicted to be within        about 3Å of the CDR's (or the effective antigen-binding site) in        a three-dimensional immunoglobulin model and capable of        interacting with the antigen or with the CDR's of the parent or        FR-patched immunoglobulin;    -   (4) have amino acid conservatively similar in structure to amino        acid at corresponding FR position of the parent immunoglobluin        predicted to be within about 3Å of the CDR's (or the effective        antigen-binding site) in a three-dimensional immunoglobulin        model and capable of interacting with the antigen or with the        CDR's of the parent or FR-patched immunoglobulin.

Each of the heavy and light chains of the FR-patched immunoglobulin willtypically comprise FR's sourced from one or more human or primateimmunoglobulins according to any one or all of the selection criteria.The FR-patched heavy and light chains of the present invention, whencombined into an intact antibody, antibody fragment, or antibody-basedderivatives (for example single-chain antibody, diabodies, etc.), willbe substantially non-immunogenic in humans and retain substantially thesame affinity and properties (for example internalization upon binding)as the parent immunoglobulin to the antigen. These affinity levelsshould vary within the range of 4-fold, and preferably within about 2fold of the parent immunoglobulin's original affinity to the antigen.

Similar principles apply to re-engineer, or patch, parentimmunoglobulins of one species with the FR's from a different species.People skilled in the art of protein and/or molecular engineering willbe able to adopt the design and principle of the present invention toproduce FR-patched immunoglobulins, or derivatives thereof. Oncedesigned, there exist a variety of techniques in constructing theFR-patched immunoglobulin sequence, for example, by site-directedmutagenesis, and gene-synthesis. The assembled FR-patched sequences willbe subcloned into expression vectors containing the appropriateimmunoglobulin constant heavy and light chains for transfection inproducer cell lines. Different cell systems can be used for theproduction of the FR-patched immunoglobulins, including bacterial,yeast, insect, and mammalian cells.

Alternatively, the immunoglobulins can be produced in the milks oftransgenic or transomatic animals, or as stored proteins in transgenicplants. The present invention offers an improved and novel methods, thatare relatively easy (no need to identify important FR amino acidinteracting with the CDR's) and highly flexible (freedom to match, andchange if necessary, individual FR's) in generating immunoglobulins withreduced or eliminated immunogenicities without sacrificing bindingaffinity and the likelihood of introducing new T- and B-cell epitopesresulting from the introduction of parent immunoglobulin's frameworkamino acids into the human FR's. The FR-patched antibodies will besuitable for human use in treating a variety of disease, either usedsingly or repeatedly, at low (less than 10 mg/m²) or high (more than 100mg/m²) doses, in naked forms or as fusion or chemical conjugates, usedalone, or in conjunction with other immunoglobulins or treatmentmodalities.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1A and FIG. 1B. Amino acid sequences (single-letter code) of theheavy chain (VH)(A) and light chain (VL)(B) variable regions of themurine anti-CD22 antibody, RFB4. CDR's are boxed. (SEQ ID NO. 33 and 34)

FIG. 2A and FIG. 2B. Comparison of the compartmentalized frameworksequences (FR's) of the heavy chain (A) and light chain (B) variableregions of RFB4, with the different human FR's of the highest homology.The FR1, FR2, FR3, and FR4 are indicated. The CDR's are boxed. Thebracketed italic next on the left of the FR sequence indicates thesource of the human FR. Amino acids in the human FR's that are differentfrom that of the corresponding murine FR's are in bold. (SEQ ID NO.35-46)

FIG. 3A and FIG. 3B. The final designed sequences (single-letter code)of the heavy chain (A) and light chain (B) variable regions of theFR-patched antibody, hpRFB4. CDR's are boxed. Amino acids in the humanFR's that are different from that of the original murine FR's are inbold. (SEQ ID NO. 47-48)

FIG. 4. Predicted SDS-PAGE analysis of purified cRFB4 and hpRFB4 underboth reducing and non-reducing conditions.

FIG. 5. Predicted flow cytometry analyses on the binding specificity andaffinity of cRFB4 and hpRFB4 on Raji cells.

FIG. 6. Predicted competition binding assay comparing the bindingaffinity between cRFB4 and hpRFB4. An irrelevant antibody was used as acontrol.

FIG. 7A and FIG. 7B. Amino acid sequences (single-letter code) of theheavy chain (A) and light chain (B) variable regions of the murineanti-CD20 antibody, 1F5. CDR's are boxed. (SEQ ID NO. 49-50)

FIG. 8A and FIG. 8B. Comparison of the compartmentalized frameworksequences (FR's) of the heavy chain (A) and light chain (B) variableregions of 1F5 with the different human FR's of the highest homology.The FR1, FR2, FR3, and FR4 are indicated. The CDR's are boxed. Thebracketed italic next on the left of the FR sequence indicates thesource of the human FR. Amino acids in the human FR's that are differentfrom that of the corresponding murine FR's are in bold. (SEQ ID NO.51-67)

FIG. 9A and FIG. 9B. The final designed sequences (single-letter code)of the heavy chain (A) and light chain (B) variable regions of theFR-patched antibody, hp1F5. CDR's are boxed. Amino acids in the humanFR's that are different from that of the original murine FR's are inbold. Murine FR's not replaced by human sequences are underlined. (SEQID NO. 68-69)

FIG. 10. Amino acid sequence of an alternative design of FR-patchedvariable regions for 1F5 (Alternative Design). CDR's are boxed. Humanframework amino acids that differ from that of the corresponding murineframeworks are in bold. A. The heavy chain variable region (VH) aminoacid sequence of FR-patched 1F5 (Alternative Design); (SEQ ID NO. 70) B.The light chain variable region (VL) amino acid sequence of FR-patched1F5 (Alternative Design). (SEQ ID NO. 71)

DETAILED DESCRIPTION OF THE INVENTION

The present invention aims to establish novel approaches in the designof immunoglobulin with high degree of homology to human or primatesequences through a process named “framework (FR) patching”. TheFR-patched immunoglobulin (patched immunoglobulin thereafter) will havesubstantially reduced, or eliminated immunogenicity when used in human,and carry most or all of the characteristics of a human immunoglobulinsuch as the ability to target specific antigens, and effector functions(for example, complement fixation, ADCC, etc.), while maintaining thespecificity and affinity of the parent immunoglobulin against a specificantigen. The patched immunoglobulin will comprise a heavy and lightchain, of which, the respective variable region will contain sequencesrepresenting FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, according toKabat's classification (Kabat et al., op. cit.). At least one of thefour FR's of each parent immunoglobulin chain containing one or morecomplementary determining regions (CDR's) will be replaced, or “patched”with, a corresponding human or primate FR. When two or more FR's of theparent immunoglobulin chain are to be replaced, they can be patched withcorresponding FR's either from the same human or primate immunoglobulin,or from different human or primate immunoglobulin within the samesubgroup or in different subgroups, or from a combination of human andprimate immunoglobulins. The patched immunoglobulins will be expressedin appropriate host system for large-scale production at typicalpharmaceutical margins, and used in humans at appropriate formats orcombinations to treat or detect a wide range of human diseases.

To ensure a better understanding of the present invention, severaldefinitions are set forth. As used herein, an “immunoglobulin” refers toa protein consisting of one or more polypeptides substantially encodedby immunoglobulin genes. A typical immunoglobulin protein contains twoheavy chains paired with two light chains. A full-length immunoglobulinheavy chain is about 50 kD in size (approximately 446 amino acids inlength), and is encoded by a heavy chain variable region gene (about 116amino acids) and a constant region gene. There are different constantregion genes encoding heavy chain constant region of different isotypessuch as alpha, gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon, and musequences. A full-length immunoglobulin light chain is about 25 Kd insize (approximately 214 amino acids in length), and is encoded by alight chain variable region gene (about 110 amino acids) and a kappa orlambda constant region gene. Naturally occurring immunoglobulin is knownas antibody, usually in the form of a tetramer consisting of twoidentical pairs of immunoglobulin chains, each pair having one light andone heavy chain. In each pair, the light and heavy chain variableregions are together responsible for binding to an antigen, and theconstant regions are responsible for the effector functions typical ofan antibody.

Immunoglobulin may be in different forms, either naturally occurring,chemically modified, or genetically-engineered, such as Fv (Huston etal., Proc. Natl. Acad. Sci. USA. 85:5879-5833; Bird et al., Science242:423-426, 1988), diabodies, mini-antibodies, Fab, Fab', F(ab')₂,bifunctional hybrid antibodies (Lanzavecchia et al., Eur. J. Immunol.17:105, 1987) (See, generally, Hood et al., “Immunology”, Benjamin, NY,2^(nd) ed. 1984; Hunkapiller and Hood, Nature 323:15-16, 1986).

The variable region of both the heavy and light chain is divided intosegments comprising four framework sub-regions (FR1, FR2, FR3, and FR4),interrupted by three stretches of hypervariable sequences, or thecomplementary determining regions (CDR's), as defined in Kabat'sdatabase (Kabat et al., op. cit.), with the CDR1 positioned between FR1and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Withoutspecifying the particular sub-regions as FR1, FR2, FR3 or FR4, aframework region, as referred by others, represents the combined FR'swithin the variable region of a single, naturally occurringimmunoglobulin chain. As used herein, a FR represents one of the foursub-regions, and FR's represents two or more of the four sub-regionsconstituting a framework region. The sequences of the framework regionsof different light or heavy chains are relatively conserved within aspecies. The framework region of an antibody is the combined frameworkregions of the constituent light and heavy chains and serves to positionand align the CDR's. The CDR's are primarily responsible for forming thebinding site of an antibody conferring binding specificity and affinityto an epitope of an antigen.

Parent antibody is an antibody of a particular species, for example,murine, which is to be re-engineered, re-shaped, or in this invention,FR-patched into a form, sequence, or structure, appropriate for use in adifferent species, for example, human, with reduced or minimizedimmunogenicity.

Chimeric antibodies are antibodies whose variable regions are linked,without significant sequence modifications from the parent V-regionsequences, to the corresponding heavy and light chain constant regionsof a different species. Construction of a chimeric antibody is usuallyaccomplished by ligating the DNA sequences encoding the variable regionsto the DNA sequences encoding the corresponding constant chains. Themost common types of chimeric antibodies are those containing murinevariable regions and human constant regions. In this case, the expressedhybrid molecule will have the binding specificity and affinity of theparent murine antibody, and the effector functions of a human antibody.Most importantly, ⅔ of the amino acids of the recombinant protein are ofhuman origin, a reduced or insignificant immunogenicity is thereforeexpected when used in human, as in the case of the therapeutic chimericantibody C2B8 (or RITUXIMAB) (Davis et al., J. Clin. Oncol.17:1851-1857, 1999; Coiffier et al., Blood 92:1927-1932, 1998;McLaughlin et al., J. Clin. Oncol. 16:2825-2833, 1998).

A “humanized” immunoglobulin is generally accepted as an immunoglobulincomprising a human framework region and one or more CDR's from anon-human immunoglobulin (Jones et al., op. cit; Verhoeyen et al., op.cit; Riechmann et al., op. cit.). The non-human immunoglobulin providingthe CDR's is called the “donor” and the human immunoglobulin providingthe framework is called the “acceptor”. Usually, as has been used andreferred to by others, an acceptor is derived from a single humanimmunoglobulin species. To maintain the affinity of the “humanized”immunoglobulin, donor amino acid residues may have to be incorporated inthe framework region of the acceptor immunoglobulin. There is a set ofcriteria for selecting a limited number of amino acids within theacceptor immunoglobulin for conversion into donor sequences, aspublished in a series of publications (U.S. Pat. No. 5,85,089; U.S. Pat.No. 5,693,762; U.S. Pat. No. 5,693,761; incorporated herein byreference). The humanized immunoglobulins may or may not containconstant regions. A humanized heavy chain immunoglobulin is a humanizedimmunoglobulin comprising a corresponding human heavy chain constantregion, and a humanized light chain immunoglobulin is a humanizedimmunoglobulin comprising a corresponding human light chain constantregion. A humanized antibody is an antibody comprising a humanized lightchain and a humanized heavy chain immunoglobulin.

A successful humanized antibody will have to have the followingcharacteristics:

-   -   (1) significantly reduced, and preferably eliminated,        immunogenicity resulting from the humanized sequences, allowing        multiple injection of the humanized antibody for human uses;    -   (2) minimally perturbed immunoreactivity including specificity        and affinity (within 3-fold) against the original antigen;    -   (3) capable of inducing human effector functions such as        complement fixation, complement-mediated cytotoxicity,        antibody-dependent cell cytotoxicity, etc.

Direct grafting of donor CDR's onto human acceptor framework withoutfurther sequence modifications, will likely result in substantial lossof antigen affinity. Although the introduction of selected donor aminoacids to acceptor framework regions will somehow rectify the problem,and most of the time, improve affinity, however, the approach istedious, requiring sometimes the assistance of computer modeling inidentifying the appropriate framework amino acid to mutate, and lackflexibility in the choice of acceptor human frameworks in an all-or-nonemode. Most importantly, it is likely to introduce potential newimmunogenic epitopes by retaining parent “donor” residues in the human“acceptor” framework.

The present invention addresses these problems and creates a novelapproach with increased flexibility and simplicity in generating aFR-patched antibody that is not immunogenic or is low in immunogenicity,yet having retained most or all of the original affinity against aspecific antigen, as in the parent antibody. Since most of the immuneresponses against a chimeric or humanized immunoglobulin will bedirected against epitopes in the variable regions, without intending tobe bound by theory, the principle by which the invention comes aboutwill be illustrated by, but not limited to, using the variable region asthe example.

There exist at least two kinds of epitopes contributing to theimmunogenicity against a protein. The so-called “T cell epitopes” areshort peptide sequences released during the degradation of proteinswithin cells and subsequently presented by molecules of the majorhistocompatability complexes (MHC) in order to trigger the activation ofT cells. For peptides presented by MHC class II, such activation of Tcells can then give rise to an antibody response by direct stimulationof B cells to produce such antibodies. A detailed analysis of thestructure of a humanized variable region reveals the unavoidableexistence of stretches of potentially immunogenic CDR's. These CDR'sphysically and functionally compartmentalize the rest of the frameworksequences into four sub-regions, namely, the FR1, FR2, FR3, and FR4(Kabat et al., op. cit.). Since T cell epitopes are linear continuousshort peptides, the presence or absence of such epitopes in each FRcompartments should have no correlation to each other, whether thedifferent FR's are derived from the same or different frameworks. Theintroduction of donor framework residues to the acceptor frameworkregion using the humanization approach of Queen et al. (U.S. Pat. No.5,85,089; U.S. Pat. No. 5,693,762; U.S. Pat. No. 5,693,761; incorporatedherein by reference) will have the possibility of generating new,immunogenic T cell epitopes, resulting in the elicitation of immuneresponses against the humanized antibody, particularly antibodyresponses against the idiotypic region formed by the donor CDR loops. Itis uncommon to have between 3 to 7 donor amino acids incorporated intoeach humanized immunoglobulin chain, greatly increasing the chances ofemergency for new T cell epitopes.

Similarly, these donor-derived residues embedded within the humanframework can form new immunogenic B-cell epitopes recognizable byantibodies. While it is well-established that re-introduction of donorresidues to the acceptor framework is important in maintaining theoriginal antigen affinity of the humanized immunoglobulin, ideally, itwould be preferable if humanization can be accomplished by directgrafting of donor CDR's onto acceptor framework without additionalmodification and loss of affinity.

The present invention provides a new approach in reducing or eliminatingthe immunogenicity of immunoglobulins whose affinity against thespecific antigen is maintained within three fold of its original level.The approach is flexible, versatile, simple, and does not usuallyrequire sophisticated computer modeling analysis (although it does notpreclude its being used). It deals with the problem of reciprocalrelation between reducing immunogenicity and maintaining affinity inhumanizing an antibody with the previous and available methodologies.Using an immunoglobulin variable region as example, a set of criteriaand principles will be followed in FR-patching the sequence. Thecriteria may be used singly, or when necessary in combination, toachieve reduced or eliminated immunogenicity, and the desired affinityor other characteristics.

In humanizing an immunoglobulin variable region by FR-patching, theparent immunoglobulin amino acid sequences are compartmentalized intoFR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 according to the classificationof Kabat et al. (opt. cit.). Each of the compartmentalized FR's will bedealt with separately and used to align with the corresponding FRsegments found in all databases, available either in the public domain,commercial entities, or private possession (for example the Kabatdatabase, opt. cit.; the National Biomedical Research Foundation ProteinIdentification Resource). An immunoglobulin can be patched with FR'sfrom more than one immunoglobulin sources. Preferably, human FR segmentswith the highest degree of homology (>60%) to the corresponding parentFR's will be used. However, amino acids in the FR's adjacent to one ormore of the 3 CDR's in the primary sequence of the immunoglobulin chainmay interact directly with the antigen (Amit et al., Science,233:747-753, 1986, which is incorporated herein by reference) andselecting these amino acids identical to the human FR's with lesserhomology will be used according to the criteria set forth below.

A human FR1 will be used when it has the highest homology to the parentFR1, preferably 100%, at three or more amino acids immediately adjacentto CDR1.

A human FR2 will be used when it has the highest homology to the parentFR2, preferably 100%, at three or more amino acids at both endsimmediately adjacent to the flanking CDR1 and CDR2.

A human FR3 will be used when it has the highest homology to the parentFR3, preferably 100%, at three or more amino acids at both endsimmediately adjacent to the flanking CDR2 and CDR3.

A human FR4 will be used when it has the highest homology to the parentFR4, preferably 100%, at three or more amino acids immediately adjacentto CDR3.

In case human FR's with 100% homology at three or more amino acidsadjacent to the CDRs cannot be identified, FR's with the closesthomology at these positions containing conservatively similar aminoacids, such as, gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr;lys, arg; and phe, tyr; will be selected.

Preferably, human FR's whose amino acids at positions known to be closeto, or have interactions with the CDR's/antigen binding site (Chothiaand Lesk, J. Mol. Biol. 196:901, 1987; Chothia et al., Nature 342:877,1989, and Tramontano et al., J. Mol. Biol. 215:175, 1990, all of whichare incorporated herein by reference), either based on computer modeling(see Levy et al., Biochemistry 28:7168-7175, 1989; Bruccoleri et al.,Nature 335:564-568, 1988; Chothia et al., Science 233:755-758, 1986, allof which are incorporated herein by reference), crystal structure,published information, or prior experience, which are identical, orconservatively similar to that of the parent FR's will be selected.

FR-patching does not preclude the introduction of parent amino acids atcorresponding positions of a patched FR where necessary, or theinclusion of FR's in the immunoglobulin from different species such asdifferent primates, murine, etc, when available databases fail toproduce a satisfactory FR that meet the above criteria. The primary goalis to produce antibodies with reduced, preferably, eliminatedimmunogenicity without substantial loss of affinity. The approachincreases the chances of success in this regard, with significantimprovements over other methods in terms of flexibility, simplicity, andease of operation.

FR-patched antibodies carrying human constant sequences will be able toinduce human immune effector functions such as complement-mediatedcytotoxicity (CM) or anti-body-dependent cellular cytotoxicity (ADCC),upon binding to the target antigens. Moreover, when injected in humanfor therapeutic or diagnostic purposes, antibodies patched with humanFR's are expected to be non-immunogenic, i.e., will not elicit antibodyresponses against the injected protein, allowing for multiple injectionsinto human patients if necessary for achieving maximum clinicalbenefits. Non-human antibodies have been reported to have significantlyshorter circulation half-lives than that of human antibodies (Shaw etal., J Immunol. 138:4534-4538, 1987). The patched antibodies, carryingmostly human sequences, will presumably have an extended half-lifereminiscent to naturally occurring human antibodies.

In the construction of a FR-patched immunoglobulin, sequence design forthe variable regions of the immunoglobulin will be done using thecriteria and principles illustrated above. The designed FR-patchedvariable region sequence will be assembled using a variety of standardrecombinant techniques well known to those skilled in the art.Preferably, the designed sequence, usually of a size of about 350 basepairs, will be gene-synthesized (Leung et al., Molecular Immunol.32:1413-1427, 1995; Daugherty et al., Nucl. Acid Res. 19:2471-2476;DeMartino et al., Antibody Immunoconj. Radiopharmaceut. 4:829, 1991;Jones et al., op. cit., all of which are incorporated herein byreference), or the individual FR's can be introduced to replace theparent FR's by methods of site- or oligonucleotide-directed mutagenesis(Gillman and Smith, Gene 8:81-97, 1979; and Roberts et al., Nature328:731-734; both of which are incorporated herein by reference).

The DNA segment encoding the FR-patched immunoglobulin will be joined toDNA sequences encoding the human heavy and light chain regions in DNAexpression vectors suitable for bacterial propagation and expression indifferent host cells. There are a variety of DNA vectors suitable forexpression in a variety of host cell systems. Appropriate DNA vectorscan be chosen for the expression of the FR-patched immunoglobulins.Typically, a suitable expression control DNA sequence is linked operablyto DNA segments encoding the immunoglobulin chains. Preferably, theexpression control sequences will be eukaryotic promoter systems invectors capable of transforming or transfecting eukaryotic host cells,but control sequences for prokaryotic hosts may also be used. Thesequence encoding the FR-patched heavy and light immunoglobulin chainscan be incorporated into one single DNA expression vector, or intoseparate heavy and light chain expression vectors. In the latter case,host cells will have to be simultaneously incorporated with both vectorsin order to produce a FR-patched antibody with the properly paired heavyand light chain polypeptides. In general, a leader sequence allowing thetransportation of the immunoglobulin polypeptide into the Golgiapparatus for later secretion is included at the N-terminal end of eachimmunoglobulin chain for expression in eukaryotic host cells. Once thevector has been incorporated into the appropriate host, the host ismaintained under conditions suitable for high level expression of thenucleotide sequences, and, as desired, the collection and purificationof the humanized light chains, heavy chains, light/heavy chain dimers orintact antibodies, binding fragments, single chain antibody (sFv),diabodies, or derivatives thereof, or other immunoglobulin forms mayfollow (see Beychok, Cells of Immunoglobulin Synthesis, Academic Press,NY, 1979, which is incorporated herein by reference).

It is a well-known fact that there are different human constant regionsfor the heavy and light chains. A particular isotype will have specificeffector characteristics that can be chosen for use for differentpurposes. Human constant region DNA sequences can be isolated inaccordance with well known procedures from a variety of human cells, butpreferably immortalized B-cells (see, Kabat op. cit. and WP87/02671).The CDR's for producing the immunoglobulins of the present inventionwill be similarly derived from monoclonal antibodies capable of bindingto the predetermined antigens, such as CD22 and CD20, for example, andproduced by well known methods in any convenient mammalian sourceincluding, mice, rat, rabbits, or other vertebrate, capable of producingantibodies. Suitable source cells for the constant region and frameworkDNA and secretion, can be obtained from a number of sources such as theAmerican Type Culture Collection (“Catalogue of Cell Lines andHybridomas,” sixth edition, 1988, Rockville, Md., USA, which isincorporated herein by reference).

DNA expression vectors containing the coding sequences for theFR-patched immunoglobulin chains operably linked to an expressioncontrol sequence (including promoter and enhancers) are typicallyreplicable in the host organisms either as episomes or as an integralpart of the host chromosomal DNA. Selectable markers such astetracycline, neomycin, beta-lactamase, etc., are included in the vectorto allow detection of cells transformed with the DNA vectors (see, forexample, U.S. Pat. No. 4,704,362, which is incorporated herein byreference).

Bacterial hosts are suitable for propagating the DNA vectors as well asexpressing the incorporated immunoglobulin DNA. For example, E. coli isthe most commonly used prokaryotic host used for cloning the DNAsequence for the present invention. Other microbial hosts that areuseful for the same purposes include, as examples, bacilli (for exampleBacillu subtilus), and other enterobacteriaceae (for example Salmonella,Serratia), and various Pseudomonas species. Expression of clonedsequences in these hosts require the presence of expression controlsequences compatible with the host cell (for example an origin ofreplication), and functional promoters to be included in the DNA vector.Example of well-known promoter system include, but not limited to,tryptophan (trp) promoter system, beta-lactamase promoter system, phagelambda promoter system, etc. These promoters are responsible forcontrolling expression, or transcription, of the functional genesequence downstream of the promoter system, which contains, in additionto all necessary motifs, and optionally with an operator sequence,ribosome binding site sequences and the like, necessary fortranscription initiation and translation.

Similarly, other microbes, such as yeast, may also be used forexpression. For example, a preferred host will be Saccharomyces, whichis a suitable host for expression vectors containing the appropriateexpression control elements, such as promoters, including3-phosphoglycerate kinase or other glycolytic enzymes, and an origin ofreplication, termination sequences and the like as desired.

Eukaryotic host cells of invertebrate origin can be used. For example,insect cells, such as hi-5, SF9, SF21. Appropriate expression vectorscontaining convenient cloning sites, promoters, termination sequences,etc., that are important for high-level expression in the host cells areavailable commercially (Invitrogen, San Diego, Calif.).

Preferably, mammalian tissue cell culture may be used to express andproduce the polypeptides of the present invention (see, Winnacker, “FromGenes to Clones,” VCH Publisher, NY, N.Y., 1987, which is incorporatedherein by reference). The most commonly used mammalian host cells areChinese Hamster Ovary (CHO) cell lines, various COS cell lines, HeLacells, and myeloma cell lines such as SP2/0 cell lines, NS0 cell lines,YB2/0 cell lines, etc, and transformed B-cells or hybridomas. These celllines are capable of conferring the right glycosylation at appropriatesite such as amino acid 297 at the heavy chain CH2 domain, and secretingfull-length immunoglobulins, and are the host cell system of choice forthis particular invention. Similar to expression vectors for other hostcells, a eukaryotic cell expression vector will contain the appropriateexpression control sequences including promoter (for example, thosederived from immunoglobulin genes, metallothionine gene, SV40,Adenovirus, cytomegalovirus, Bovine Papilloma Virus, and the like),enhancers, usually with a leader sequence for directing the expressedpolypeptide to the Golgi apparatus for glycosylation and export, the DNAsegments of interest (for example, the heavy and light chain encodingsequences and expression control sequences), a termination codon, othernecessary processing information sites (such as ribosome binding sites,RNA splice sites, a polyadenylation sequence, and transcriptionalterminator sequences), and a selection marker (such as mutant Dhfr,glutamine synthetase (GS), hygromycin, neomycin) (see Kellems, “GeneAmplification in mammalian cells”, Marcel Dekker Inc., NY, N.Y., 1993;which is incorporated herein by reference).

There exist a plethora of established and well-known methods forintroducing the vectors containing the DNA segments of interest into thehost cell, either transiently or stably integrated into the host cellgenome. They include, but not limited to, calcium chloride transfection,calcium phosphate treatment, electroporation, lipofection, etc. (See,Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Press, 1982; which is incorporated herein by reference).Identification of host cells incorporated with the appropriateexpression vector will be achievable typically by first growing cellsunder selection pressure in accordance with the selectable marker usedin the vector, and detection of secreted proteins, for example, thewhole antibodies containing two pairs of heavy and light chains, orother immunoglobulin forms of the present invention, by standardprocedures such as ELISA and Western analysis. Purification of theexpressed immunoglobulin can be purified according to standardprocedures of the art, including ammonium sulfate precipitation,affinity columns, column chromatography, gel electrophoresis and thelike (see, generally, R. Scopes, “Protein Purification”,Springer-Verlag, NY, 1982). Substantially pure immunoglobulins of atleast about 90 to 95% homogeneity are preferred, and 98 to 99% or morehomogeneity most preferred, for pharmaceutical uses. Once purified,partially or to homogeneity as desired, the polypeptides may then byused therapeutically (including extracorporeally) or in developing andperforming assay procedures, immunofluorescent stainings, and the like(See, generally, Immunological Methods Vols I and II, Lefkovits andPernis, eds., Academic Press, New York, N.Y., 1979 and 1981).

The antibodies of the present invention will typically find useindividually, or in combination with other treatment modalities, intreating diseases susceptible to antibody-based therapy. For example,the immunoglobulins can be used for passive immunization, or the removalof unwanted cells or antigens, such as by complement mediated lysis, allwithout substantial adverse immune reactions (for example anaphylacticshock) associated with many prior antibodies.

A preferable usage of the antibodies of the present invention will bethe treatment of diseases using their naked forms (naked antibodies) atdosages ranging from 50 mg to 400 mg/m², administered either locally atthe lesion site, subcutaneously, intravenously, and intramuscularly,etc. Multiple dosing at different intervals will be performed to achieveoptimal therapeutic or diagnostic responses, for example, at weeklyintervals, once a week, for four weeks. Usage of the antibodies derivedfrom the present invention can be combined with different treatmentmodalities, such as chemotherapeutic drugs (for example CHOP, Dox, 5-Fu,. . . etc), radiotherapy, radioimmunotherapy, vaccines, enzymes,toxins/immunotoxins, or other antibodies derived from the presentinvention or others. The antibodies of the present invention, ifspecific for the idiotype of an anti-tumor antibody, can be used astumor vaccines for the elicitation of Ab3 against the tumor antigen.Numerous additional agents, or combinations of agents, well-known tothose skilled in the art may also be utilized.

Additionally, the antibodies of the present invention can be utilized indifferent pharmaceutical compositions. The antibodies can be used intheir naked forms, or as conjugated proteins with drugs, radionuclides,toxins, cytokines, soluble factors, hormones, enzymes (for examplecarboxylesterase, ribonuclease), peptides, antigens (as tumor vaccine),DNA, RNA, or any other effector molecules having a specific therapeuticfunction with the antibody moiety serving as the targeting agents ordelivery vehicles. Moreover, the antibodies or antibody derivatives,such as antibody fragments, single-chain Fv, diabodies, etc. of thepresent invention can be used as fusion proteins to other functionalmoieties, such as, antibodies or antibody derivatives of a differentinvention (for example as bispecific antibodies), toxins, cytokines,soluble factors, hormones, enzymes, peptides, etc. Differentcombinations of pharmaceutical composition, well-known to those skilledin the art may also be utilized.

FR-patched antibodies of the present invention can also be used for invitro purposes, for example, as diagnostic tools for the detection ofspecific antigens, or the like.

The following examples are offered by way of illustration, not bylimitation.

EXPERIMENTAL

In designing the amino acid sequence of the FR-patched immunoglobulinchain, the murine variable region sequence (applies to both VH and VL)was compartmentalized into FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4,according to Kabat's classification (Kabat et al., op. cit.). Selectionof the individual FR's for patching was in accordance to the guidelinesas described previously.

A human FR1 will be used when it has the highest homology to the parentFR1, preferably 100%, at three or more amino acids immediately adjacentto CDR1.

A human FR2 will be used when it has the highest homology to the parentFR2, preferably 100%, at three or more amino acids at both endsimmediately adjacent to the flanking CDR1 and CDR2.

A human FR3 will be used when it has the highest homology to the parentFR3, preferably 100%, at three or more amino acids at both endsimmediately adjacent to the flanking CDR2 and CDR3.

A human FR4 will be used when it has the highest homology to the parentFR4, preferably 100%, at three or more amino acids immediately adjacentto CDR3.

In case human FR's with 100% homology at three or more amino acidsadjacent to the CDRs cannot be identified, FR's with the closesthomology at these positions containing conservatively similar aminoacids, such as, gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr;lys, arg; and phe, tyr; will be selected.

Preferably, human FR's whose amino acids at positions known to be closeto, or have interactions with the CDR's/antigen binding site (Chothiaand Lesk, op. cit.; Chothia et al., op. cit., and Tramontano et al., op.cit.), either based on computer modeling (see Levy et al., Biochemistryop. cit.; Bruccoleri et al., op. cit.; Chothia et al., op. cit.),crystal structure, published information, or prior experience, which areidentical, or conservatively similar to that of the parent FR's will beselected.

In case where a particular human FR satisfying all the above isunavailable, and direct patching results in the loss of affinity orspecificity, murine residues considered to have interactions with theantigen binding site, or contribute to the final affinity of theantibody, will be introduced back to the best available FR.Alternatively, the particular FR with no matching human counterpart willbe retained and used in its murine composition without modification; thefinal FR-patched sequence will contain a mixture of human and murineFR's. For the purpose of illustration, two murine anti-B cell antibodieswill be FR-patched using the approach as described herein in thisinvention.

EXAMPLE 1 FR-Patched Anti-CD22 Antibody Design of Genes for FR-PatchedAnti-CD22 Light and Heavy Chain

The heavy and light chain sequence of a murine anti-CD22 antibody, RFB4(Li et al., Cell Immunol. 118:85, 1989; Mansfield et al., Blood90:2020-2026, 1997) is used as an example to illustrate the approach ofusing FR-patching to reduce or eliminate immunogenicity of there-engineered antibody. The sequences of the heavy (a) and light chain(b) variable region for the murine antibody are shown in FIG. 1.

Patching of the individual FRs for the heavy chain variable region forRFB4 was done as follows:

-   -   a. FR1: the FR1 sequence of the murine VH was compared with the        FR1 sequences of human VH from the Kabat's database (Kabat et        al., op. cit.). Although human FR1 of the highest sequence        homology is preferred, particular emphasis on the sequence        closest to the CDR1 was taken. There are three FR1 sequences        that are of high homology to the murine FR1. They are, namely,        EIK, RF-SJ1, and WAS. The FR1 with the highest overall homology        with the five residues closest to the CDR1 identical to the        murine parent is EIK, however, there is a missing residue in        position 12, which can create potential problems affecting the        immunoreactivity of the resultant antibody. The preferred FR1        picked for the patching was therefore from WAS. First, except at        position 28, the third closest residue to CDR1, a whole stretch        of 11 amino acids next to the CDR1 is identical to the murine        parent. In position 28, a serine residue is found instead of an        alanine in the murine sequence. Since serine is considered as a        hydroxylated version of alanine, the change is conservative.        Moreover, residues that are different between the human and        murine are relatively similar in characteristics. For example,        valine and leucine in position 5, lysine and glutamine at        position 13, lysine and arginine at position 19, and alanine and        serine at position 28. Therefore, human sequence from WAS was        chosen for patching the FR1 of the anti-CD22 antibody (FIG. 2A).    -   b. FR2: by the same token and based on the degree of homology,        the human WAS sequence is chosen for patching the FR2 of the        anti-CD22 antibody (FIG. 2A).    -   c. FR3: with the sequences closest to the CDR2, and CDR3 being        identical, and the high degree of homology, the human GAL        sequence was selected for patching the FR3 of the anti-CD22        antibody (FIG. 2A).    -   d. FR4: there are many human FR4 with the sequence closest to        the CDR3 being identical, and a high degree of homology to the        murine parent. In this example, the human DOB sequence was        selected for patching the FR4 of the anti-CD22 antibody (FIG.        2A).

The final design of the FR-patched VH sequence (FIG. 3A) for theanti-CD22 antibody is composed of the human WAS FR1 and FR2, and the GALFR3 and DOB FR4, replacing the original VH FR's of the anti-CD22antibody. There is no single mutation or re-introduction of murine FRresidues in the final design of the FR-patched sequence.

Using a similar strategy, the sequence design for the FR-patched lightchain (VL) was done as follows:

-   -   a. FR1: human JOH was chosen for patching the FR1 of the murine        VL. It has a high degree of sequence homology and the stretch of        8 amino acids adjacent to the CDR1 being identical to the parent        sequence (FIG. 2B).    -   b. FR2: human Vd'CL was chosen for patching the FR2 of the        murine VL, for similar reasons. More than 4 identical sequences        are adjacent to the CDR1, and CDR2 (FIG. 2B).    -   c. FR3: human WES was chosen for patching the FR3 of the murine        VL. FR3 has the longest sequence, and the sequence homology        between WES and the murine FR3 is high, with the sequences        flanking the CDR2 and CDR3 being identical (FIG. 2B).    -   d. FR4: human RZ was chosen for patching the FR4 of the murine        VL, for similar reasons (FIG. 2B).

The final design of the FR-patched VL sequence (FIG. 3B) for theanti-CD22 antibody is composed of the human JOH FR1, Vd'CL FR2, WES FR3,and RZ FR4, replacing the original VL FR's of the anti-CD22 antibody.Once again, there is no single mutation or re-introduction of murine FRresidues in the final design of the FR-patched sequence.

Construction of the FR-Patched Heavy and Light Chain Genes

The designed heavy and light chain variable region sequences of theFR-patched antibody are assembled by a combination of oligonucleotidesynthesis and PCR using a variety of published methods (Leung et al.,op. cit.; Daugherty et al., op. cit.; DeMartino et al., op. cit.; Joneset al., op. cit.).

To construct the FR-patched heavy chain variable region sequence (SEQ IDno. 1), the full DNA sequence is divided into two halves: the N-terminalhalf and the C-terminal half. Both are constructed separately by PCR andthe complete variable region sequence is formed by joining the N- andC-terminal halves at the KpnI site.

The N-terminal half is constructed as follows: a N-template (SEQ ID no.3) is a synthetic sense-strand oligonucleotide (111-mer) encoding aminoacids 14-50 of the VH region (SEQ ID no. 2). The template isPCR-amplified by two primers:

The 5′ Primer (SEQ ID no. 4) is a synthetic sense-strand oligonucleotide(57-mer) encoding amino acids 1-19 of the VH region. The 3′ end of theprimer overlaps with the 5′ end of the template by 18 nucleotides.

The 3′ Primer (SEQ ID no. 5) is a synthetic anti-sense-strandoligonucleotide (48-mer) encoding amino acids 43-59. The primer overlapswith the template by 21 nucleotides.

The N-template (SEQ ID no. 3) is PCR-amplified using the 5′ and 3′primer set (SEQ ID no. 4 & 5) using standard techniques and procedures.

The C-terminal half is constructed as follows: a C-template (SEQ ID no.6) is a synthetic sense-strand oligonucleotide (132-mer) encoding aminoacids 68-111 of the VH region (SEQ ID no. 2). The template isPCR-amplified by two primers:

The 5′ Primer (SEQ ID no. 7) is a synthetic sense-strand oligonucleotide(60-mer) encoding amino acids 55-74 of the VH region. The 3′ end of theprimer overlaps with the 5′ end of the template by 21 nucleotides.

The 3′ Primer (SEQ ID no. 8) is a synthetic anti-sense-strandoligonucleotide (58-mer) encoding amino acids 105-123 of the VH region.The primer and the template overlap by 21 nucleotides.

The C-template (SEQ ID no. 6) is PCR-amplified using the 5′ and 3′primer set (SEQ ID no. 7 & 8) using standard techniques and procedures.

For the construction of the full-length FR-patched RFB4 VH domain, theN-template (SEQ ID no. 3, 111-mer), C-template (SEQ ID no. 6, 132-mer),and their respective 5′- and 3′ primers (SEQ ID no. 4 & 5 forN-template, and SEQ ID no. 7 & 8 for C-template), are synthesized on anautomated Applied Biosystem 380B DNA synthesizer (Foster City, Calif.).The oligonucleotides are desalted by passing through a CHROMOSPIN-10™column (Clonetech, Palo Alto, Calif.). The oligonucleotides are adjustedto a final concentration of 20 μM. One μl of template oligonucleotidesat various dilutions (10×, 100×, 1000× and 10000×, etc.) are mixed with5 μl of their corresponding flanking primers in the presence of 10 μl of10×PCR Buffer (500 mM KCl, 100 mM Tris.HCl buffer, pH 8.3, 15 mM MgCl2)and 5 units of AMPLITAQ™ DNA polymerase (Perkin Elmer, AppliedBiosystems Division, Foster City, Calif.). This reaction mixture isadjusted to a final volume of 100 μl and subjected to 30 cycles of PCRreaction consisting of denaturation at 94° C. for 1 minute, annealing at50° C. for 1 minutes, and polymerization at 72° C. for 1 minute. The PCRreaction mixtures are analyzed under 2% agarose gel electrophoresis. Thehighest template dilution that gives rise to sufficiently abundantproduct of the right size will be chosen for further processing.

Double-stranded PCR-amplified products for the N- and C-templates aregel-purified, restriction-digested with KpnI. The restricted N- andC-double stranded DNA are ligated at the KpnI site, and the ligatedproducts are subjected to another round of PCR amplification using the5′ primer for the N-template (SEQ ID no. 4) and the 3′ primer for theC-template (SEQ ID no. 8). The PCR product with a size of ˜350 isdirectly cloned into the TA cloning vector (Invitrogen, San Diego,Calif.). The sequence of the cloned fragment is confirmed by Sanger'smethod (Sanger et al., PNAS 74:5463-5467, 1977) to be identical to thedesigned VH sequence. The confirmed sequence is used to replace the VHsequence of a heavy chain expression vector containing an IgH promoter,an Ig enhancer, a human IgG1 constant region genomic sequence, and aselectable marker, gpt. The final heavy chain expression vector isdesignated as hpRFB4pSMh.

To construct the FR-patched light chain variable region sequence (SEQ IDno. 9), the full length VL variable region sequence is divided into twohalves. The N-terminal and C-terminal halves are assembled separately byPCR and joined together via the SpeI site.

The N-terminal half is constructed as follows: a N-template (SEQ ID no.11) is a synthetic sense-strand oligonucleotide (108-mer) encoding aminoacids 11-46 of the VL region (SEQ ID no. 10). The template isPCR-amplified by two primers:

The 5′ Primer (SEQ ID no. 12) is a synthetic sense-strandoligonucleotide (51-mer) encoding amino acids 1-17 of the VL region. The3′ end of the primer overlaps with the 5′ end of the template by 21nucleotides.

The 3′ Primer (SEQ ID no. 13) is a synthetic anti-sense-strandoligonucleotide (40-mer) encoding amino acids 40-53. The primer overlapswith the template by 18 nucleotides.

The N-template (SEQ ID no. 11) is PCR-amplified using the 5′ and 3′primer set (SEQ ID no. 12 & 13) using standard techniques andprocedures.

The C-terminal half is constructed as follows: a C-template (SEQ ID no.14) is a synthetic sense-strand oligonucleotide (120-mer) encoding aminoacids 59-98 of the VL region (SEQ ID no. 10). The template isPCR-amplified by two primers:

The 5′ Primer (SEQ ID no. 15) is a synthetic sense-strandoligonucleotide (49-mer) encoding amino acids 50-65 of the VL region.The 3′ end of the primer overlaps with the 5′ end of the template by 21nucleotides.

The 3′ Primer (SEQ ID no. 16) is a synthetic antisense-strandoligonucleotide (48-mer) encoding amino acids 92-107 of the VL region.The primer and the template overlap by 21 nucleotides.

The C-template (SEQ ID no. 14) is PCR-amplified using the 5′ and 3′primer set (SEQ ID no. 15 & 16) using standard techniques andprocedures.

For the construction of the FR-patched RFB4 VL domain, the N-template(SEQ ID no. 11, 108-mer), C-template (SEQ ID no. 14, 120-mer), and theirrespective 5′- and 3′ primers (SEQ ID no. 12 & 13 for N-template, andSEQ ID no. 15 & 16 for C-template), are synthesized on an automatedApplied Biosystem 380B DNA synthesizer. The oligonucleotides aredesalted by passing through a CHROMOSPIN-10™ column (Clonetech). Theoligonucleotides are adjusted to a final concentration of 20 μM. One μlof template oligonucleotides at various dilutions (10×, 100×, 1000× and10000×, etc.) are mixed with 5 μl of their corresponding flankingprimers in the presence of 10 μl of 10×PCR Buffer (500 mM KCl, 100 mMTris.HCl buffer, pH 8.3, 15 mM MgCl2) and 5 units of AMPLITAQ™ DNApolymerase (Perkin Elmer). This reaction mixture is adjusted to a finalvolume of 100 μl and subjected to 30 cycles of PCR reaction consistingof denaturation at 94° C. for 1 minute, annealing at 50° C. for 1minutes, and polymerization at 72° C. for 1 minute. The PCR reactionmixtures are analyzed under 2% agarose gel electrophoresis. The highesttemplate dilution that gives rise to sufficiently abundant product ofthe right size will be chosen for further processing.

Double-stranded PCR-amplified products for the N- and C-templates aregel-purified, restriction-digested with SpeI. The restricted N- andC-double stranded DNA are ligated at the SpeI site, and the ligatedproducts are subjected to another round of PCR-amplification using the5′ primer for the N-template (SEQ ID no. 12) and the 3′ primer for theC-template (SEQ ID no. 16). The PCR product with a size of ˜320 isdirectly cloned into the TA cloning vector (Invitrogen). The sequence ofthe cloned fragment is confirmed by Sanger's method (Sanger op. cit.) tobe identical to the designed VL sequence. The confirmed sequence is usedto replace the VL sequence of a light chain expression vector containingan IgH promoter, an Ig enhancer, a human kappa constant region genomicsequence, and a selectable marker, hyg. The final light chain expressionvector is designated as hpRFB4pSMk.

Expression and Affinity of FR-Patched Antibody

The expression plasmids hpRFB4pSMh and hpRFB4pSMk are linearized andco-transfected into mouse Sp2/0 cells. Cells transfected with theplasmids are selected in the presence of mycophenolic acid and/orhygromycin B conferred by the gpt and hyg genes on the plasmids bystandard methods. Cells surviving selection are tested for humanantibody secretion using ELISA methods. Clones that are identified to besecreting human antibody are expanded for production in 500 ml rollerbottles. Antibodies are purified using standard protein A columns. Thepurified antibody is analyzed in a SDS-PAGE gel under both reducing andnon-reducing conditions (Predicted results shown in FIG. 4). Theaffinity of the FR-patched antibody (hpRFB4) is first evaluated by flowcytometry. Raji cells (5×10⁵) are incubated with 1 μg of either purifiedhpRFB4 or chimeric RFB4 (cRFB4) in a final volume of 100 μl of PBSsupplemented with 1% FCS and 0.01% (w/v) sodium azide (PBS-FA). cRFB4differs from hpRFB4 in the variable region sequences which are deriveddirectly from the murine parent without modifications. The mixtures areincubated for 30 minutes at 4° C. and washed three times with PBS toremove unbound antibodies. The binding levels of the antibodies to Rajicells are assessed by the addition of a 20× diluted FITC-labeled, goatanti-human IgG1, Fc fragment-specific antibodies (JacksonImmunoResearch, West Grove, Pa.) in a final volume of 100 μl in PBS-FA,and incubating for 30 minutes at 4° C. The mixture is washed three timeswith PBS and fluorescence intensities are measured by a FACSCANfluorescence-activated cell sorter (Becton Dickinson, Bedford, Mass.)(Predicted results shown in FIG. 5) The predicted results indicate thatboth antibodies bind well to Raji cells with similar affinity.

To compare the affinity of the antibody before and after re-engineeringthe VH and VL sequences of RFB4, a competitive binding assay isperformed. Fixed amount (10× dilution from stock) of FITC-conjugatedRFB4 (Ancell Corporation, Bayport, Minn.) is mixed with varyingconcentrations of either cRFB4 or hpRFB4. The mixtures are added to Rajicells in a final volume of 100 μl in PBS-FA, and incubated for 30minutes at 4° C. After washing three times with PBS, the fluorescenceintensities of Raji cells bound with the FITC-RFB4 are measured byFASCAN (Becton Dickinson, Bedford, Mass.). The predicted resultsindicate that FR-patching of the RFB4 sequence does not have significanteffects on the affinity of the re-engineered antibody (Predicted resultsshown in FIG. 6).

EXAMPLE 2 FR-Patched Anti-CD20 Antibody Design of Genes for FR-PatchedAnti-CD20 Light and Heavy Chain.

The heavy and light chain sequence of a murine anti-CD20 antibody, 1F5(Shan et al. 1999. J Immunol. 162:6589-6595) is used as an example toillustrate the approach of using FR-patching to reduce or eliminateimmunogenicity of the re-engineered antibody. The sequences of the heavyand light chain variable region for the murine antibody are shown inFIG. (7).

In designing the amino acid sequence of the FR-patched immunoglobulinfor 1F5, the same set of rules as described previously applies. However,there are always situations when no appropriate FR's fulfill all theabove-mentioned requirements. The FR-patching approach offers a greatdegree of flexibility allowing the introduction of murine residues inthe problematic FR's, or alternatively, inclusion of the original murineFR's without modifications. The resultant FR-patched antibody willpresumably have significantly reduced immunogenicity compared to amurine or chimeric antibody. An anti-CD20 antibody, 1F5, is used as anexample for FR-patching to illustrate these points.

Patching of the individual FR's of the 1F5 VH sequence was done asfollows:

-   -   a. FR1: the FR1 sequence of the murine VH was compared with the        FR1 sequences of human VH from the Kabat's database (Kabat et        al., op. cit.). Human FR1 of the highest sequence homology is        preferred, particularly at the sequences closest to the CDR1.        The human FR1 sequence from LS2'′CL has close to 80% of sequence        homology to that of the murine anti-CD20 antibody, and the 10        residues adjacent to the CDR1 are identical to the murine parent        sequence. Therefore, the human FR1 sequence from LS2'CL was        chosen for patching the FR1 of the anti-CD20 antibody (FIG. 8A)    -   b. FR2: the FR2 sequence of the human NEWM was chosen for        patching the FR2 sequence of the anti-CD20 antibody. It should        be noted that although the third residue of the NEWM FR2 closest        to the CDR1 is not identical to that of the murine parent        sequence, it is a conserved K to R conversion (FIG. 8A).    -   c. FR3: human heavy chain FR3 sequences with satisfactorily high        sequence homology and identical sequences adjacent to the CDR2        and CDR3 could not be identified. Although the human FR3 from        783C'CL exhibited 78% of sequence homology, the residues        flanking the CDR2 are drastically different, despite the        differences being conserved. For example, the K, A, and L at        positions 57, 58 and 60 (Kabat's numbering, Kabat et al., op.        cit.) which are the 1^(st), 2^(nd), and 4^(th) residues closest        to the CDR2 are replaced by the conserved human residues R, V        and I, respectively. Nevertheless, the high number of changes in        proximity to the CDR2, albeit conservative, could result in        significant conformational changes at the antigen binding site.        Without risking the loss of affinity, and as an illustration on        the flexibility of the FR-patching approach, the FR3 would not        be patched with any of the human FR's. Instead, the murine FR3        sequence was retained in this particular antibody (FIG. 8A).    -   d. FR4: there are many human FR4 with the sequence closest to        the CDR3 being identical, and a high degree of homology to the        murine parent. In this example, the human 4G12'CL sequence was        selected for patching the FR4 of the anti-CD20 antibody (FIG.        8A).

The final design of the FR-patched VH sequence (FIG. 9A) for theanti-CD20 antibody is composed of the human LS2'CL FR1, NEWM FR2, murine1F5 FR3 and 4G12'CL FR4, replacing the original VH FR's of the murineanti-CD20 antibody.

An alternative design will be a patched VH containing the murine CDRsembedded in human LS2'CL FR1, NEWM FR2, 783C'CL FR3, and 4G12'CL FR4(FIG. 10A). For the purpose of illustration, the construction of theformer version will be described below.

Using a similar strategy, the sequence design for the FR-patched lightchain was constructed as follows:

-   -   a. FR1: human BJ19 was chosen for patching the FR1 of the murine        VL. This is the human FR1 sequence with the highest homology to        the murine parent (61%). Moreover, some of the human residues        that are different from that of the murine are conserved. For        example, the E to D, and K to R conversions at positions 18 and        19, respectively, are conserved changes (FIG. 8B).    -   b. FR2: although there is a human FR2, MOT, that was found to be        of high sequence homology (73%) to the murine FR2, the        Tryptophan at position 32 (Kabat's numbering, Kabat et al., op.        cit.), the 3^(rd) residues closest to the CDR2, was replaced by        a non-conservative Valine in the MOT FR2 sequence. This        replacement potentially might have a significant effect on the        final conformation of the antigen binding site. It was therefore        determined that the murine FR2 of the VL domain would remain in        the design of the FR-patched antibody (FIG. 8B).    -   c. FR3: human WES was chosen for patching the FR3 of the murine        VL. FR3 has the longest sequence, and the sequence homology        between WES and the murine FR3 is 71%, with the three human        residues flanking the CDR2 and CDR3 being identical to that of        the murine (FIG. 8B).    -   d. FR4: human λ FR4 sequence from NIG-58 was chosen for patching        the FR4 of the murine VL, for similar reasons. The sequences are        72% homologous to the stretch of 7 residues adjacent to the CDR3        being identical between the human and murine (FIG. 8B).

The final design of the FR-patched VL sequence (FIG. 9B) for theanti-CD20 antibody is composed of the human BJ19 FR1, murine 1F5 FR2,WES FR3, and NIG-58 FR4, replacing the original VL FR's of the anti-CD20antibody. An alternative design of FR-patched VL will be composed of thehuman BJ19 FR1, MOT FR2, WES FR3, and NIG-58 FR4, forming the scaffoldsupporting the CDR loops (FIG. 10B). For the purpose of illustration inthis application, only the construction of the former FR-patched VL willbe described below.

Construction of the FR-Patched Heavy and Light Chain Genes

The designed heavy and light chain variable region sequences of theFR-patched antibody are assembled by a combination of oligonucleotidesynthesis and PCR using a variety of published methods.

To construct the FR-patched heavy chain variable region sequence (SEQ IDno. 17), the full DNA sequence is divided into two halves. TheN-terminal half and the C-terminal half are constructed separately byPCR and the complete variable region sequence is formed by joining theN- and C-terminal halves at the SpeI site.

The N-terminal half is constructed as follows: a N-template (SEQ ID no.19) is a synthetic sense-strand oligonucleotide (1 14-mer) encodingamino acids 12-49 of the VH region (SEQ ID no. 18). The template isPCR-amplified by two primers:

The 5′ Primer (SEQ ID no. 20) is a synthetic sense-strandoligonucleotide (57-mer) encoding amino acids 1-19 of the VH region. The3′ end of the primer overlaps with the 5′ end of the template by 24nucleotides.

The 3′ Primer (SEQ ID no. 21) is a synthetic anti-sense-strandoligonucleotide (55-mer) encoding amino acids 43-60. The primer overlapswith the template by 21 nucleotides.

The N-template (SEQ ID no. 19) is PCR-amplified using the 5′ and 3′primer set (SEQ ID no. 20 & 21) using standard techniques andprocedures.

The C-terminal half is constructed as follows: a C-template (SEQ ID no.22) is a synthetic sense-strand oligonucleotide (126-mer) encoding aminoacids 70-111 of the VH region (SEQ ID no. 18). The template isPCR-amplified by two primers:

The 5′ Primer (SEQ ID no. 23) is a synthetic sense-strandoligonucleotide (61-mer) encoding amino acids 57-76 of the VH region.The 3′ end of the primer overlaps with the 5′ end of the template by 21nucleotides.

The 3′ Primer (SEQ ID no. 24) is a synthetic antisense-strandoligonucleotide (59-mer) encoding amino acids 105-123 of the VH region.The primer and the template overlap by 21 nucleotides.

The C-template (SEQ ID no. 22) is PCR-amplified using the 5′ and 3′primer set (SEQ ID no. 23 & 24) using standard techniques andprocedures.

For the construction of the FR-patched 1F5 VH domain, the N-template(SEQ ID no. 19, 114-mer), C-template (SEQ ID no. 22, 126-mer), and theirrespective 5′- and 3′ primers (SEQ ID no. 20 & 21 for N-template, andSEQ ID no. 23 & 24 for C-template), are synthesized on an automatedApplied Biosystem 380B DNA synthesizer. The oligonucleotides aredesalted by passing through a CHROMOSPIN-10™ column (Clonetech). Theoligonucleotides are adjusted to a final concentration of 20 μM. One μlof template oligonucleotides at various dilutions (10×, 100×, 1000× and10000×, etc.) are mixed with 5 μl of their corresponding flankingprimers in the presence of 10 μl of 10×PCR Buffer (500 mM KCl, 100 mMTris.HCl buffer, pH 8.3, 15 mM MgCl₂) and 5 units of AMPLITAQ™ DNApolymerase (Perkin Elmer). This reaction mixture is adjusted to a finalvolume of 100 μl and subjected to 30 cycles of PCR reaction consistingof denaturation at 94° C. for 1 minute, annealing at 50° C. for 1.5minutes, and polymerization at 72° C. for 1 minute. The PCR reactionmixtures are analyzed under 2% agarose gel electrophoresis. The highesttemplate dilution that gives rise to sufficiently abundant product ofthe right size will be chosen for further processing.

Double-stranded PCR-amplified products for the N- and C-templates aregel-purified, restriction-digested with KpnI site. The N- and C-doublestranded DNA are ligated at the SpeI site, and the ligated products aresubjected to another round of PCR amplification using the 5′ primer forthe N-template (SEQ ID no. 19) and the 3′ primer for the C-template (SEQID no. 22). The PCR product with a size of ˜350 is directly cloned intothe TA cloning vector (Invitrogen). The sequence of the cloned fragmentis confirmed by Sanger's method (Sanger et al. op. cit.) to be identicalto the designed VH sequence. The confirmed sequence is used to replacethe VH sequence of a heavy chain expression vector containing an IgHpromoter, an Ig enhancer, a human IgG1 constant region genomic sequence,and a selectable marker, gpt. The final heavy chain expression vector isdesignated as hp1F5pSMh.

To construct the FR-patched light chain variable region sequence (SEQ IDno. 25), the full length VL variable region sequence is divided into twohalves. The N-terminal and C-terminal halves are assembled separately byPCR and joined together via the BspEI site.

The N-terminal half is constructed as follows: a N-template (SEQ ID no.27) is a synthetic sense-strand oligonucleotide (129-mer) encoding aminoacids 9-51 of the VL region (SEQ ID no. 26). The template isPCR-amplified by two primers:

The 5′ Primer (SEQ ID no. 28) is a synthetic sense-strandoligonucleotide (45-mer) encoding amino acids 1-15 of the VH region. The3′ end of the primer overlaps with the 5′ end of the template by 21nucleotides.

The 3′ Primer (SEQ ID no. 29) is a synthetic anti-sense-strandoligonucleotide (40-mer) encoding amino acids 45-57. The primer overlapswith the template by 21 nucleotides.

The N-template (SEQ ID no. 27) is PCR-amplified using the 5′ and 3′primer set (SEQ ID no. 28 & 29) using standard techniques andprocedures.

The C-terminal half is constructed as follows: a C-template (SEQ ID no.30) is a synthetic sense-strand oligonucleotide (120-mer) encoding aminoacids 61-100 of the VH region (SEQ ID no. 26). The template isPCR-amplified by two primers:

The 5′ Primer (SEQ ID no. 31) is a synthetic sense-strandoligonucleotide (43-mer) encoding amino acids 54-67 of the VH region.The 3′ end of the primer overlaps with the 5′ end of the template by 21nucleotides.

The 3′ Primer (SEQ ID no. 32) is a synthetic antisense-strandoligonucleotide (42-mer) encoding amino acids 94-107 of the VH region.The primer and the template overlap by 21 nucleotides.

The C-template (SEQ ID no. 30) is PCR-amplified using the 5′ and 3′primer set (SEQ ID no. 31 & 32) using standard techniques andprocedures.

For the construction of the FR-patched 1F5 VL domain, the N-template(SEQ ID no. 27, 129-mer), C-template (SEQ ID no. 30, 120-mer), and theirrespective 5′- and 3′ primers (SEQ ID no. 28 & 29 for N-template, andSEQ ID no. 31 & 32 for C-template), are synthesized on an automatedApplied Biosystem 380B DNA synthesizer. The oligonucleotides aredesalted by passing through a CHROMOSPIN-10™ column (Clonetech). Theoligonucleotides are adjusted to a final concentration of 20 μM. One μlof template oligonucleotides at various dilutions (10×, 100×, 1000× and10000×, etc.) are mixed with 5 μl of their corresponding flankingprimers in the presence of 10 μl of 10×PCR Buffer (500 mM KCl, 100 mMTris.HCl buffer, pH 8.3, 15 mM MgCl₂) and 5 units of AMPLITAQ™ DNApolymerase (Perkin Elmer). This reaction mixture is adjusted to a finalvolume of 100 μl and subjected to 30 cycles of PCR reaction consistingof denaturation at 94° C. for 1 minute, annealing at 50° C. for 1.5minutes, and polymerization at 72° C. for 1 minute. The PCR reactionmixtures are analyzed under 2% agarose gel electrophoresis. The highesttemplate dilution that gives rise to sufficiently abundant product ofthe right size will be chosen for further processing.

Double-stranded PCR-amplified products for the N- and C-templates aregel-purified, restriction-digested with SpeI site. The N- and C-doublestranded DNA are ligated at the BspEI site, and amplified using the 5′primer for the N-template (SEQ ID no. 12) and the 3′ primer for theC-template (SEQ ID no. 16). The PCR product with a size of ˜320 isdirectly cloned into the TA cloning vector (Invitrogen). The sequence ofthe cloned fragment is confirmed by Sanger's method (Sanger et al., op.cit.) to be identical to the designed VL sequence. The confirmedsequence is used to replace the VL sequence of a light chain expressionvector containing an IgH promoter, an Ig enhancer, a human kappaconstant region genomic sequence, and a selectable marker, hyg. Thefinal light chain expression vector is designated as hp1F5pSMk.

1. A method for producing an immunoglobulin containing 3 heavy chain and3 light chain complementary determining regions from the mouse 1F5immunoglobulin and framework sequences from human immunoglobulinscomprising the steps of: a. dividing the framework sequences from the1F5 immunoglobulin into compartmentalized sub-regions of FR1, FR2, FR3,and FR4 according to the classification of the Kabat Database; b.comparing the individual sub-region framework sequences to correspondingsequences in a collection of human immunoglobulin chains; c. selecting,from the collection, the appropriate human framework sequences toreplace the original framework sequences of the FR1, FR2, FR3 and FR4sub-regions of the 1F5 immunoglobulin, wherein the resultingimmunoglobulin comprises a heavy chain variable region having the aminoacid sequence of SEQ ID NO: 70, and a light chain variable region havingthe amino acid sequence of SEQ ID NO: 71; d. assembling the frameworksequences selected in step c; e. subcloning the assembled frameworksequences of step d into heavy and light chain expression vectorscontaining appropriate immunoglobulin constant heavy and light chainnucleotide sequences; f. co-transfecting mouse Sp2/0 cells with theexpression vectors of step e; g. culturing the mouse Sp2/0 cells of stepf under conditions permitting expression and secretion ofimmunoglobulin.
 2. The method of claim 1 wherein the immunoglobulinconstant heavy and light chain nucleotide sequences are human IgG1 andhuman kappa constant region sequences, respectively.